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采用细胞融合技术筛选抗19-去甲睾酮(nortestosterone,NT)单克隆抗体(monoclonal antibody,mAb)杂交瘤细胞株,体内诱性腹水法制备NT mAb,建立异源性间接竞争ELISA(indirect competitive ELISA,icELISA)标准曲线,并对其特异性、理化因素的影响和基质效应性进行探讨.结果表明,筛选的5株杂交瘤细胞分泌的单抗腹水效价为0.64×105～2.56×105,半数抑制浓度IC50为0.55～1.0ng/mL,抗体为IgG1亚型,具k轻链,亲和常数Ka为2.6×109～4.7×109L/mol.建立的icELISA标准曲线在磷酸盐缓冲液(phosphate buffered saline,PBS)中的线性检测范围为0.004～85.8ng/mL,IC50值为0.55ng/mL,检测限(limit of detection,LOD)为0.002ng/mL;抗体与17a-去甲睾酮的交叉反应率(cross-reactivity,CR)为62%,与其他化合物无CR.标准品稀释液中甲醇的最佳含量为10%,牛尿1:20倍稀释可消除基质干扰,ELISA方法和液质联用检测(LC-MS/MS)的相关性良好(R2=0.9871).
The monoclonal antibody (mAb) monoclonal antibody against 19-nortestosterone (NT) was screened by cell fusion technique and the NT mAb was prepared by in vivo ascitic fluid ascites assay. The indirect competitive ELISA , icELISA) standard curve, and its specificity, physicochemical factors and matrix effects were discussed.The results showed that the screening of five hybridoma cells secreted monoclonal antibody ascites titer of 0.64 × 105 ~ 2.56 × 105, half The inhibitory concentration (IC50) was 0.55 ~ 1.0ng / mL, the antibody was IgG1 subtype, with kappa light chain and affinity constant Ka of 2.6 × 109 ~ 4.7 × 109L / mol.The established icELISA standard curve was expressed in phosphate buffered saline, PBS) with a linear range of 0.004-85.8 ng / mL, an IC50 value of 0.55 ng / mL and a limit of detection (LOD) of 0.002 ng / mL. The antibody cross-reactivity with 17a- The cross-reactivity (CR) of 62% was compared with that of other compounds without CR. The optimum concentration of methanol in the standard diluent was 10%, and the 1:20 dilution of bovine urine could eliminate matrix interference. ELISA and LC- Correlation with test (LC-MS / MS) was good (R2 = 0.9871).